The goal of this project is to determine the molecular mechanisms involved in the assembly of the triad junction between T-tubules and sarcoplasmic reticulum during the development of excitation-contraction (E-C) coupling in skeletal muscle. Visualizing the T-tubules in vitro with a fluorescent lipid probe and with antibodies showed that T-tubules form independently from the plasma membrane and that an internal precursor of the T-system exists prior to its connection with the plasma membrane. Light and electron microscopic immunocytochemistry of the skeletal muscle dihydropyridine (DHP) receptor (the putative voltage sensor in E-C coupling) revealed the colocalization of the alphal and alpha2 subunits in the junctional membranes of the T-tubules. The membrane cytoskeletal protein ankyrin was also localized in the triad. Immunofluorescence localization of the DHP receptor alpha subunits in developing normal muscle in culture showed that the subunits cluster in receptor-rich domains of the developing T-system (presumably forming triads), but in dysgenic (mdg) myotubes the alphal subunit is absent and the alpha2 subunit fails to aggregate in the T-tubules. Both the expression of alphal and the normal distribution patterns of alphal and alpha2 subunits can be locally restored by the fusion of dysgenic myotubes with normal cells. This suggests that a specific protein-protein interaction of the alpha subunits of the DHP receptor is involved in the normal organization of the receptor complex.